Differential knockout of an allele of a heterozygous ELANE gene-II

Abstract

Methods for inactivating in a cell a mutant allele of the elastase, neutrophil expressed gene (ELANE gene) gene having a mutation associated with severe congenital neutropenia (SCN) or cyclic neutropenia (CyN) and which cell is heterozygous at one or more polymorphic sites selected from the group consisting of: rs10424470, rs4807932, rs10414837, rs376107533, rs3761010, rs10413889, rs3761007, rs10409474, rs3761005, rs351107, rs3761001, rs740021, rs781452480, rs371057361, rs570466264, rs1041904080, rs7250194, rs17216649, rs199720952, rs6510983, rs17223066, rs7255385, rs112639467, rs141213775, rs28591229, rs10469327, rs3834645, rs1683564, rs71335276, and rs8107095, the method comprising introducing to the cell a composition comprising: a CRISPR nuclease or a sequence encoding the CRISPR nuclease; and a first RNA molecule comprising a guide sequence portion having 17-30 nucleotides, wherein a complex of the CRISPR nuclease and the first RNA molecule affects a double strand break in the mutant allele of the ELANE gene the method optionally further comprising introduction of a second RNA molecule comprising a guide sequence portion capable of complexing with a CRISPR nuclease, wherein the complex of the second RNA molecule and CRISPR nuclease affects a second double strand break in the ELANE gene.

The invention claimed is: 
     
       1. A method for inactivating in a cell a mutant allele of the elastase, neutrophil expressed gene (ELANE gene) having a mutation associated with severe congenital neutropenia (SCN) or cyclic neutropenia (CyN) and which cell is heterozygous at the polymorphic site rs1683564, the method comprising introducing to the cell a composition comprising:
 a Cas9 CRISPR nuclease or a sequence encoding said CRISPR nuclease; and 
 a first RNA molecule comprising a guide sequence portion having 17-30 nucleotides, 
 
       wherein the guide sequence portion of the first RNA molecule comprises 17-30 nucleotides which comprise 17-20 contiguous nucleotides set forth in any one of SEQ ID NOs: 335-354; and 
       wherein a complex of the CRISPR nuclease and the first RNA molecule affects a double strand break in the mutant allele of the ELANE gene. 
     
     
       2. The method of  claim 1 , wherein the CRISPR nuclease is an spCas9 nuclease and wherein the complex of the CRISPR nuclease and the first RNA molecule affects a double strand break in the mutant allele of the ELANE gene. 
     
     
       3. The method of  claim 1 , further comprising introduction of a second RNA molecule and a Cas9 CRISPR nuclease or a sequence encoding the Cas 9 CRISPR nuclease, wherein the complex of the second RNA molecule and the CRISPR nuclease affects a second double strand break in the ELANE gene, and wherein the second RNA molecule comprises a guide sequence portion having 17-30 nucleotides guide sequence which comprise 17-20 contiguous nucleotides set forth in SEQ ID NO: 428. 
     
     
       4. The method of  claim 1 , comprising obtaining the cell with an ELANE gene mutation associated with SCN or CyN from a subject with an ELANE gene mutation related to SCN or CyN and/or suffering from SCN or CyN. 
     
     
       5. The method of  claim 4 , comprising obtaining the cell from the subject by mobilization and/or by apheresis or by bone marrow aspiration. 
     
     
       6. The method of  claim 4 , wherein the cell is exposed ex vivo to one or more human cytokines prior to introducing the composition to the cell. 
     
     
       7. The method of  claim 4 , wherein the cell is culture expanded to obtain cells, and wherein the cells are cultured with one or more of: stem cell factor (SCF), interleukin-3 (“IL-3”), and granulocyte-macrophage colony-stimulating factor (“GM-CSF”);
 wherein the cells are cultured with at least one cytokine; and/or 
 wherein the at least one cytokine is a recombinant human cytokine.